Heterogeneity in IgG-CD16 signaling in infectious disease outcomes.

In this review, we discuss how IgG antibodies can modulate inflammatory signaling during viral infections with a focus on CD16a-mediated functions. We describe the structural heterogeneity of IgG antibody ligands, including subclass and glycosylation that impact binding by and downstream activity of CD16a, as well as the heterogeneity of CD16a itself, including allele and expression density. While inflammation is a mechanism required for immune homeostasis and resolution of acute infections, we focus here on two infectious diseases that are driven by pathogenic inflammatory responses during infection. Specifically, we review and discuss the evolving body of literature showing that afucosylated IgG immune complex signaling through CD16a contributes to the overwhelming inflammatory response that is central to the pathogenesis of severe forms of dengue disease and coronavirus disease 2019 (COVID-19).

Longitudinal study comparing IgG antibodies induced by heterologous prime-boost COVID-19 vaccine.

Vaccines are critical cost-effective tools to control the COVID-19 pandemic. The heterologous prime-boost vaccination has been used by many countries to overcome supply issues, so the effectiveness and safety of this strategy need to be better clarified. This study aims to verify the effect of heterologous prime-boost COVID-19 vaccination on healthcare professionals from Dante Pazzanese Hospital in Brazil. It was performed serological assays of vaccinated individuals after 2-dose of CoronaVac (Sinovac; n = 89) or ChAdOx1 nCoV-19 (Oxford-AstraZeneca; n = 166) followed by a BNT162b2 booster (Pfizer-BioNTech; n = 255). The serum antibodies anti-S (spike), anti-N (nucleocapsid), and anti-RBD (receptor binding domain) were assessed by enzyme-linked immunosorbent assay. The heterologous booster dose induced a 10-fold higher anti-Spike antibody regardless of the 2-dose of a prime vaccine. It was strikingly observed that BNT162b2 enhanced levels of anti-spike antibodies, even in those individuals who did not previously respond to the 2-dose of CoronaVac. In conclusion, the heterologous scheme of vaccination using mRNA as a booster vaccine efficiently enhanced the antibody response against SARS-CoV-2, especially benefiting those elderly who were seronegative with a virus-inactivated vaccine.

Impact of BNT162b2 mRNA anti-SARS-CoV-2 vaccine on interferon-alpha production by plasmacytoid dendritic cells and autoreactive T cells in patients with systemic lupus erythematosus: The COVALUS project.

OBJECTIVE: To evaluate the specific response of SLE patients to BNT162b2 vaccination and its impact on autoimmunity defined as in vivo production of interferon-alpha (IFNα) by plasmacytoid dendritic cells (pDCs) and autoreactive immune responses. METHODS: Our prospective study included SLE patients and healthy volunteers (HV) who received 2 doses of BNT162b2 vaccine 4 weeks apart. Subjects under immunosuppressive drugs or with evidence of prior COVID-19 were excluded. IgG anti-Spike SARS-CoV-2 (anti-S) antibodies, anti-S specific-B cells, anti-S specific T cells, in vivo INF-α production by pDCs, activation marker expression by pDCs and autoreactive anti-nuclear T cells were quantified before first injection, before second injection, and 3 and 6 months after first injection. RESULTS: Vaccinated SLE patients produced significantly lower IgG antibodies and specific B cells against SARS-CoV-2 as compared to HV. In contrast, anti-S T cell response did not significantly differ between SLE patients and HV. Following vaccination, the surface expression of HLA-DR and CD86 and the in vivo production of IFNα by pDCs significantly increased in SLE patients. The boosted expression of HLA-DR on pDCs induced by BNT162b2 vaccine correlated with the overall immune responses against SARS-CoV-2 (anti-S antibodies: r = 0.27 [0.05-0.46], p = 0.02; anti-S B cells: r = 0.19 [-0.03-0.39], p = 0.09); anti-S T cells: r = 0.28 [0.05-0.47], p = 0.016). Eventually, anti-SARS-CoV-2 vaccination was associated with an overall decrease of autoreactive T cells (slope = - 0.00067, p = 0.015). CONCLUSION: BNT162b2 vaccine induces a transient in vivo activation of pDCs in SLE that contributes to the immune responses against SARS-CoV-2. Unexpectedly BNT162b2 vaccine also dampens the pool of circulating autoreactive T cells, suggesting that vaccination may have a beneficial impact on SLE disease.

Synthetic nanobodies as tools to distinguish IgG Fc glycoforms.

Protein glycosylation is a crucial mediator of biological functions and is tightly regulated in health and disease. However, interrogating complex protein glycoforms is challenging, as current lectin tools are limited by cross-reactivity while mass spectrometry typically requires biochemical purification and isolation of the target protein. Here, we describe a method to identify and characterize a class of nanobodies that can distinguish glycoforms without reactivity to off-target glycoproteins or glycans. We apply this technology to immunoglobulin G (IgG) Fc glycoforms and define nanobodies that specifically recognize either IgG lacking its core-fucose or IgG bearing terminal sialic acid residues. By adapting these tools to standard biochemical methods, we can clinically stratify dengue virus and SARS-CoV-2 infected individuals based on their IgG glycan profile, selectively disrupt IgG-Fcγ receptor binding both in vitro and in vivo, and interrogate the B cell receptor (BCR) glycan structure on living cells. Ultimately, we provide a strategy for the development of reagents to identify and manipulate IgG Fc glycoforms.

Immune modulation as a consequence of SARS-CoV-2 infection.

Erroneous immune responses in COVID-19 could have detrimental effects, which makes investigation of immune network underlying COVID-19 pathogenesis a requisite. This study aimed to investigate COVID-19 related alterations within the frame of innate and adaptive immunity. Thirty-four patients clinically diagnosed with mild, moderate and severe COVID-19 disease were enrolled in this study. Decreased ILC1 and increased ILC2 subsets were detected in mild and moderate patients compared to healthy controls. NK cell subsets and cytotoxic capacity of NK cells were decreased in severe patients. Moreover, CD3+ T cells were reduced in severe patients and a negative correlation was found between CD3+ T cells and D-dimer levels. Likewise, moderate and severe patients showed diminished CD3+CD8+ T cells. Unlike T and NK cells, plasmablast and plasma cells were elevated in patients and IgG and IgA levels were particularly increased in severe patients. Severe patients also showed elevated serum levels of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-8, reduced intracellular IFN-γ and increased intracellular IL-10 levels. Our findings emphasize that SARS-CoV-2 infection significantly alters immune responses and innate and acquired immunity are differentially modulated in line with the clinical severity of the disease. Elevation of IL-10 levels in NK cells and reduction of CD3+ and CD8+ T cells in severe patients might be considered as a protective response against the harmful effect of cytokine storm seen in COVID-19.

Monoclonal Antibody Engineering and Design to Modulate FcRn Activities: A Comprehensive Review.

Understanding the biological mechanisms underlying the pH-dependent nature of FcRn binding, as well as the various factors influencing the affinity to FcRn, was concurrent with the arrival of the first recombinant IgG monoclonal antibodies (mAbs) and IgG Fc-fusion proteins in clinical practice. IgG Fc-FcRn became a central subject of interest for the development of these drugs for the comfort of patients and good clinical responses. In this review, we describe (i) mAb mutations close to and outside the FcRn binding site, increasing the affinity for FcRn at acidic pH and leading to enhanced mAb half-life and biodistribution, and (ii) mAb mutations increasing the affinity for FcRn at acidic and neutral pH, blocking FcRn binding and resulting, in vivo, in endogenous IgG degradation. Mutations modifying FcRn binding are discussed in association with pH-dependent modulation of antigen binding and (iii) anti-FcRn mAbs, two of the latest innovations in anti-FcRn mAbs leading to endogenous IgG depletion. We discuss the pharmacological effects, the biological consequences, and advantages of targeting IgG-FcRn interactions and their application in human therapeutics.

Urtica dioica Agglutinin: A plant protein candidate for inhibition of SARS-COV-2 receptor-binding domain for control of Covid19 Infection.

Despite using effective drugs and vaccines for Covid 19, due to some limitations of current strategies and the high rate of coronavirus mutation, the development of medicines with effective inhibitory activity against this infection is essential. The SARS-CoV-2 enters the cell by attaching its receptor-binding domain (RBD) of Spike to angiotensin-converting enzyme-2 (ACE2). According to previous studies, the natural peptide Urtica dioica agglutinin (UDA) exhibited an antiviral effect on SARS-CoV, but its mechanism has not precisely been elucidated. Here, we studied the interaction between UDA and RBD of Spike protein of SARS-CoV-2. So, protein-protein docking of RBD-UDA was performed using Cluspro 2.0. To further confirm the stability of the complex, the RBD-UDA docked complex with higher binding affinity was studied using Molecular Dynamic simulation (via Gromacs 2020.2), and MM-PBSA calculated the binding free energy of the system. In addition, ELISA assay was used to examine the binding of UDA with RBD protein. Results were compared to ELISA of RBD-bound samples of convalescent serum IgG (from donors who recovered from Covid 19). Finally, the toxicity of UDA is assessed by using MTT assay. The docking results show UDA binds to the RBD binding site. MD simulation illustrates the UDA-RBD complex is stable during 100 ns of simulation, and the average binding energy was calculated to be -47.505 kJ/mol. ELISA and, MTT results show that UDA binds to RBD like IgG-RBD binding and may be safe in human cells. Data presented here indicate UDA interaction with S-protein inhibits the binding sites of RBD, it can prevent the virus from attaching to ACE2 and entering the host cell.

Development and evaluation of a low cost IgG ELISA test based in RBD protein for COVID-19.

Serology tests for SARS-CoV-2 have proven to be important tools to fight against the COVID-19 pandemic. These serological tests can be used in low-income and remote areas for patient contact tracing, epidemiologic studies and vaccine efficacy evaluations. In this study, we used a semi-stable mammalian episomal expression system to produce high quantities of the receptor-binding domain-RBD of SARS-CoV-2 in a simple and very economical way. The recombinant antigen was tested in an in-house IgG ELISA for COVID-19 with a panel of human sera. A performance comparison of this serology test with a commercial test based on the full-length spike protein showed 100% of concordance between tests. Thus, this serological test can be an attractive and inexpensive option in scenarios of limited resources to face the COVID-19 pandemic.

Micro-Fourier-transform infrared reflectance spectroscopy as tool for probing IgG glycosylation in COVID-19 patients.

It has been reported that patients diagnosed with COVID-19 become critically ill primarily around the time of activation of the adaptive immune response. However the role of antibodies in the worsening of disease is not obvious. Higher titers of anti-spike immunoglobulin IgG1 associated with low fucosylation of the antibody Fc tail have been associated to excessive inflammatory response. In contrast it has been also reported that NP-, S-, RBD- specific IgA, IgG, and IgM are not associated with SARS-CoV-2 viral load, indicating that there is no obvious correlation between antibody response and viral antigen detection. In the present work the micro-Fourier-transform infrared reflectance spectroscopy (micro-FTIR) was employed to investigate blood serum samples of healthy and COVID-19-ill (mild or oligosymptomatic) individuals (82 healthcare workers volunteers in "Instituto de Infectologia Emilio Ribas", São Paulo, Brazil). The molecular-level-sensitive, multiplexing quantitative and qualitative FTIR data probed on 1 µL of dried biofluid was compared to signal-to-cutoff index of chemiluminescent immunoassays CLIA and ELISA (IgG antibodies against SARS-CoV-2). Our main result indicated that 1702-1785 [Formula: see text] spectral window (carbonyl C=O vibration) is a spectral marker of the degree of IgG glycosylation, allowing to probe distinctive sub-populations of COVID-19 patients, depending on their degree of severity. The specificity was 87.5 % while the detection rate of true positive was 100%. The computed area under the receiver operating curve was equivalent to CLIA, ELISA and other ATR-FTIR methods ([Formula: see text]). In summary, overall discrimination of healthy and COVID-19 individuals and severity prediction as well could be potentially implemented using micro-FTIR reflectance spectroscopy on blood serum samples. Considering the minimal and reagent-free sample preparation procedures combined to fast (few minutes) outcome of FTIR we can state that this technology is suitable for fast screening of immune response of individuals with COVID-19. It would be an important tool in prospective studies, helping investigate the physiology of the asymptomatic, oligosymptomatic, or severe individuals and measure the extension of infection dissemination in patients.

Pre-COVID-19 Immunity to Common Cold Human Coronaviruses Induces a Recall-Type IgG Response to SARS-CoV-2 Antigens Without Cross-Neutralisation.

The capacity of pre-existing immunity to human common coronaviruses (HCoV) to cross-protect against de novo COVID-19is yet unknown. In this work, we studied the sera of 175 COVID-19 patients, 76 healthy donors and 3 intravenous immunoglobulins (IVIG) batches. We found that most COVID-19 patients developed anti-SARS-CoV-2 IgG antibodies before IgM. Moreover, the capacity of their IgGs to react to beta-HCoV, was present in the early sera of most patients before the appearance of anti-SARS-CoV-2 IgG. This implied that a recall-type antibody response was generated. In comparison, the patients that mounted an anti-SARS-COV2 IgM response, prior to IgG responses had lower titres of anti-beta-HCoV IgG antibodies. This indicated that pre-existing immunity to beta-HCoV was conducive to the generation of memory type responses to SARS-COV-2. Finally, we also found that pre-COVID-19-era sera and IVIG cross-reacted with SARS-CoV-2 antigens without neutralising SARS-CoV-2 infectivity in vitro. Put together, these results indicate that whilst pre-existing immunity to HCoV is responsible for recall-type IgG responses to SARS-CoV-2, it does not lead to cross-protection against COVID-19.

Development and Validation of an Enzyme Immunoassay for Detection and Quantification of SARS-CoV-2 Salivary IgA and IgG.

Oral fluids offer a noninvasive sampling method for the detection of Abs. Quantification of IgA and IgG Abs in saliva allows studies of the mucosal and systemic immune response after natural infection or vaccination. We developed and validated an enzyme immunoassay (EIA) to detect and quantify salivary IgA and IgG Abs against the prefusion-stabilized form of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein expressed in suspension-adapted HEK-293 cells. Normalization against total Ab isotype was performed to account for specimen differences, such as collection time and sample volume. Saliva samples collected from 187 SARS-CoV-2 confirmed cases enrolled in 2 cohorts and 373 prepandemic saliva samples were tested. The sensitivity of both EIAs was high (IgA, 95.5%; IgG, 89.7%) without compromising specificity (IgA, 99%; IgG, 97%). No cross-reactivity with endemic coronaviruses was observed. The limit of detection for SARS-CoV-2 salivary IgA and IgG assays were 1.98 ng/ml and 0.30 ng/ml, respectively. Salivary IgA and IgG Abs were detected earlier in patients with mild COVID-19 symptoms than in severe cases. However, severe cases showed higher salivary Ab titers than those with a mild infection. Salivary IgA titers quickly decreased after 6 wk in mild cases but remained detectable until at least week 10 in severe cases. Salivary IgG titers remained high for all patients, regardless of disease severity. In conclusion, EIAs for both IgA and IgG had high specificity and sensitivity for the confirmation of current or recent SARS-CoV-2 infections and evaluation of the IgA and IgG immune response.

Endothelial Cell-Activating Antibodies in COVID-19.

OBJECTIVE: While endothelial dysfunction has been implicated in the widespread thromboinflammatory complications of COVID-19, the upstream mediators of endotheliopathy remain, for the most part, unknown. This study was undertaken to identify circulating factors contributing to endothelial cell activation and dysfunction in COVID-19. METHODS: Human endothelial cells were cultured in the presence of serum or plasma from 244 patients hospitalized with COVID-19 and plasma from 100 patients with non-COVID-19-related sepsis. Cell adhesion molecules (E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 [ICAM-1]) were quantified using in-cell enzyme-linked immunosorbent assay. RESULTS: Serum and plasma from COVID-19 patients increased surface expression of cell adhesion molecules. Furthermore, levels of soluble ICAM-1 and E-selectin were elevated in patient serum and correlated with disease severity. The presence of circulating antiphospholipid antibodies was a strong marker of the ability of COVID-19 serum to activate endothelium. Depletion of total IgG from antiphospholipid antibody-positive serum markedly reduced the up-regulation of cell adhesion molecules. Conversely, supplementation of control serum with patient IgG was sufficient to trigger endothelial activation. CONCLUSION: These data are the first to indicate that some COVID-19 patients have potentially diverse antibodies that drive endotheliopathy, providing important context regarding thromboinflammatory effects of autoantibodies in severe COVID-19.

CD4+ T Cell Immune Specificity Changes After Vaccination in Healthy And COVID-19 Convalescent Subjects.

The immune response promoted by SARS-CoV-2 vaccination is relevant to develop novel vaccines and optimized prevention strategies. We analyzed the adaptive immunity in healthy donors (HD) and convalescent individuals (CD), before and after administering BNT162b2 vaccine. Our results revealed specific changes in CD4+ T cell reactivity profile in vaccinated HD and CD, with an increase in S1 and S2 positive individuals, proportionally higher for S2. On the contrary, NCAP reactivity observed in HD and CD patients was no longer detectable after vaccination. Despite the substantial antibody response in CD, MPro-derived peptides did not elicit CD4+ lymphocyte activation in our assay in either condition. HD presented an increment in anti-S and anti-RBD IgG after first dose vaccination, which increased after the second vaccination. Conversely, anti-S and anti-RBD IgG and IgA titers increased in already positive CD after first dose administration, remaining stable after second dose inoculation. Interestingly, we found a strong significant correlation between S1-induced CD4+ response and anti-S IgA pre-vaccination, which was lost after vaccine administration.

Saliva as a useful tool for evaluating upper mucosal antibody response to influenza.

Mucosal immunity plays a crucial role in controlling upper respiratory infections, including influenza. We established a quantitative ELISA to measure the amount of influenza virus-specific salivery IgA (sIgA) and salivary IgG (sIgG) antibodies using a standard antibody broadly reactive to the influenza A virus. We then analyzed saliva and serum samples from seven individuals infected with the A(H1N1)pdm09 influenza virus during the 2019-2020 flu seasons. We detected an early (6-10 days post-infection) increase of sIgA in five of the seven samples and a later (3-5 weeks) increase of sIgG in six of the seven saliva samples. Although the conventional parenteral influenza vaccine did not induce IgA production in saliva, vaccinated individuals with a history of influenza infection had higher basal levels of sIgA than those without a history. Interestingly, we observed sIgA and sIgG in an asymptomatic individual who had close contact with two influenza cases. Both early mucosal sIgA secretion and late systemically induced sIgG in the mucosal surface may protect against virus infection. Despite the small sample size, our results indicate that the saliva test system can be useful for analyzing upper mucosal immunity in influenza.

Differential Dynamics of SARS-CoV-2 Binding and Functional Antibodies upon BNT162b2 Vaccine: A 6-Month Follow-Up.

To investigate the dynamic association among binding and functional antibodies in health-care-workers receiving two doses of BNT162b2 mRNA COVID-19-vaccine, SARS-CoV-2 anti-RBD IgG, anti-Trimeric-S IgG, and neutralizing antibodies (Nabs) were measured in serum samples collected at 2 weeks, 3 months, and 6 months from full vaccination. Despite the high correlation, results for anti-RBD and anti-Trimeric S IgG were numerically different even after recalculation to BAU/mL following WHO standards indications. Moreover, after a peak response at 2 weeks, anti-RBD IgG levels showed a 4.5 and 13 fold decrease at 3 and 6 months, respectively, while the anti-Trimeric S IgG presented a less pronounced decay of 2.8 and 4.7 fold. Further different dynamics were observed for Nabs titers, resulting comparable at 3 and 6 months from vaccination. We also demonstrated that at NAbs titers ≥40, the area under the receiver operating characteristic curve and the optimal cutoff point decreased with time from vaccination for both anti-RBD and anti-Trimeric S IgG. The mutating relation among the anti-RBD IgG, anti-Trimeric S IgG, and neutralizing antibodies are indicative of antibody maturation upon vaccination. The lack of standardized laboratory procedures is one factor interfering with the definition of a correlate of protection from COVID-19.

A pandemic-enabled comparison of discovery platforms demonstrates a naïve antibody library can match the best immune-sourced antibodies.

As a result of the SARS-CoV-2 pandemic numerous scientific groups have generated antibodies against a single target: the CoV-2 spike antigen. This has provided an unprecedented opportunity to compare the efficacy of different methods and the specificities and qualities of the antibodies generated by those methods. Generally, the most potent neutralizing antibodies have been generated from convalescent patients and immunized animals, with non-immune phage libraries usually yielding significantly less potent antibodies. Here, we show that it is possible to generate ultra-potent (IC50 < 2 ng/ml) human neutralizing antibodies directly from a unique semisynthetic naïve antibody library format with affinities, developability properties and neutralization activities comparable to the best from hyperimmune sources. This demonstrates that appropriately designed and constructed naïve antibody libraries can effectively compete with immunization to directly provide therapeutic antibodies against a viral pathogen, without the need for immune sources or downstream optimization.

The Kinetics of COVID-19 Vaccine Response in a Community-Vaccinated Population.

We used a noninvasive electrochemical quantitative assay for IgG Abs to SARS-CoV-2 S1 Ag in saliva to investigate the kinetics of Ab response in a community-based population that had received either the Pfizer or Moderna mRNA-based vaccine. Samples were received from a total of 97 individuals, including a subset of 42 individuals who collected samples twice weekly for 3 mo or longer. In all, >840 samples were collected and analyzed. In all individuals, salivary SARS-CoV-2 S1 IgG Ab levels rose sharply in the 2-wk period after their second vaccination, with peak Ab levels seen at 10-20 d after vaccination. We observed that 20%, 10%, and 2.4% of individuals providing serial samples had a 90%, 95%, and 99% drop, respectively, from peak levels during the duration of monitoring, and in two patients, Abs fell to prevaccination levels (5%). The use of noninvasive quantitative salivary Ab measurement can allow widespread, cost-effective monitoring of vaccine response.

Anti-SARS-CoV-2 antibodies elicited by COVID-19 mRNA vaccine exhibit a unique glycosylation pattern.

Messenger RNA-based vaccines against COVID-19 induce a robust anti-SARS-CoV-2 antibody response with potent viral neutralization activity. Antibody effector functions are determined by their constant region subclasses and by their glycosylation patterns, but their role in vaccine efficacy is unclear. Moreover, whether vaccination induces antibodies similar to those in patients with COVID-19 remains unknown. We analyze BNT162b2 vaccine-induced IgG subclass distribution and Fc glycosylation patterns and their potential to drive effector function via Fcγ receptors and complement pathways. We identify unique and dynamic pro-inflammatory Fc compositions that are distinct from those in patients with COVID-19 and convalescents. Vaccine-induced anti-Spike IgG is characterized by distinct Fab- and Fc-mediated functions between different age groups and in comparison to antibodies generated during natural viral infection. These data highlight the heterogeneity of Fc responses to SARS-CoV-2 infection and vaccination and suggest that they support long-lasting protection differently.

SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus envelope binds to the angiotensin converting enzyme 2 (ACE2) on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.

Deep dissection of the antiviral immune profile of patients with COVID-19.

In light of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants potentially undermining humoral immunity, it is important to understand the fine specificity of the antiviral antibodies. We screened 20 COVID-19 patients for antibodies against 9 different SARS-CoV-2 proteins observing responses against the spike (S) proteins, the receptor-binding domain (RBD), and the nucleocapsid (N) protein which were of the IgG1 and IgG3 subtypes. Importantly, mutations which typically occur in the B.1.351 "South African" variant, significantly reduced the binding of anti-RBD antibodies. Nine of 20 patients were critically ill and were considered high-risk (HR). These patients showed significantly higher levels of transforming growth factor beta (TGF-ß) and myeloid-derived suppressor cells (MDSC), and lower levels of CD4+ T cells expressing LAG-3 compared to standard-risk (SR) patients. HR patients evidenced significantly higher anti-S1/RBD IgG antibody levels and an increased neutralizing activity. Importantly, a large proportion of S protein-specific antibodies were glycosylation-dependent and we identified a number of immunodominant linear epitopes within the S1 and N proteins. Findings derived from this study will not only help us to identify the most relevant component of the anti-SARS-CoV-2 humoral immune response but will also enable us to design more meaningful immunomonitoring methods for anti-COVID-19 vaccines.